All human cells are regulated by extracellular signals that are transmitted through cell surface receptors. The vast majority of these receptors constitute a superfamily of G-protein coupled receptors (GPCRs), Drugs that act on GPCRs are widely used in therapies against a large variety of human diseases, including neurological diseases such as Parkinson's disease. GPCRs are the single largest target of current drug discovery programs yet, of the approximately 200 GPCR with known functions, only a very small fraction of them are currently utilized in drug discovery due to technology limitations, leaving many of them as putative new targets for the development of new therapeutics. The majority of them signal through second messenger cAMP. Atto Bioscience has developed a novel cAMP assay for a real-time detection of cAMP in living cells using a proprietary cyclic nucleotide-gated (CNG) channel mutant. With the support of a Phase 1 grant from NINDS, we created stable cell lines and optimized assay conditions with both Ca 2+and membrane potential dyes to detect activity of the CNG channels. The CNG-cAMP technology was adapted to multiple assay platforms including FLIPR and bench-top fluorescence plate readers. This is the only commercially available high throughput screen (HTS) assay system capable of detecting receptor activations with temporal resolution. The technology has been licensed for drug discovery to pharmaceutical companies, validating both the technology and its potential market. Our goal is to develop full lines of products to enable commercialization of CNG-cAMP biosensor technology. It includes the development of additional Gs- and Gi-coupled receptor cell lines, the development of CNG cell lines in different cell types, and the development of dye reagents for the technology. We believe that this cAMP biosensor technology will not only support drug discovery, but also allow researchers to examine receptor activation and signal transduction in a new way previously not possible. The specific aims are: (1) generate, evaluate and produce another 20 Gs-coupled receptor cell lines for drug discovery and receptor biological studies; (2) develop and produce 40 Gi-coupled receptor ceil lines; (3) introduce and evaluate CNG sensor into 10 different cell lines and develop them into marketable products; (4) design, synthesize, and evaluate new membrane potential dyes, and produce sufficient reagents for 30,000,000 tests; (5) identify and test new quenchers, and develop 2 reagent kits for CNG-cAMP assay; (6) develop assay for GPCR arrays.